PROJECT SUMMARY BASIC RESEARCH PROJECT Our goal in this project is to investigate the molecular mechanisms by which the ME/CFS microbiome interacts with the immune system to cause disease. Abnormalities in immune activation are likely key contributors to ME/CFS disease severity. However, we do not know to what extent these abnormalities are: 1) microbial in origin, i.e., caused by microbial dysbiosis in ME/CFS patients; 2) caused by metabolites produced by microbiota that influence the immunological or metabolic pathophysiology; or 3) caused by genetic or environmental perturbation of immune sensitivity in ME/CFS patients. There is thus a strong need to identify the immune cells, bacteria, and molecular pathways that drive abnormal immune activation in ME/CFS. Here, we will interrogate microbes and immune cells harvested from ME/CFS patients through complementary pipelines, each designed to isolate one side of the microbe-immune axis. The microbial pipeline will compare ME/CFS- related bacteria collected from patients to bacteria from healthy controls using a set of relevant immune assays. The immune pipeline will compare immune cells collected from ME/CFS patients to healthy immune cells, exposing both to a battery of microbial triggers and immune cell activators. Together, these pipelines will pinpoint with molecular resolution the specific immune-mediated disease triggers unique to or significantly enriched in the ME/CFS condition. We expect to determine the relative contributions of microbes and immune cells toward the immune pathology observed in these patients. We also expect to identify how ME/CFS-related gut microbes impact different compartments of the immune system and to identify the immunomodulatory metabolite repertoire that underlies these interactions. This research project is highly synergistic with the clinical project, in that together they will establish microbial and immune correlates of ME/CFS disease severity and a framework for disease mechanisms. This project will also contribute to the goals of the CRC through: characterization of microbial and immune cellular and molecular mechanisms, identification of potential new therapeutic targets with characterized mechanism of action, and identification of potential microbial or transcriptional triggers, biomarkers, and risk factors. Our Specific Aims are: 1) To identify immunomodulatory bacterial strains from ME/CFS patients that may underlie immunopathology (Microbial Pipeline); and 2) To determine the response of ME/CFS patient immune cells to microbial modulators based on transcriptomic interrogation and epigenetic states (Immune Pipeline). Impact: By probing the immune responses of ME/CFS patients to microbial stimuli, we will define the molecular mechanisms by which ME/CFS-related gut microbes mediate immune activation. This work will also lay the foundation for rational discovery of therapeutics that target microbe-immune interactions, and development of engineered probiotics for ME/CFS treatment.